1. Field of the Invention
This invention relates generally to a method for aligning, compensating, and/or calibrating a multiple fluorescent channel flow cytometer for subsequent analysis of samples, and to a kit of fluorescent microbeads useful for such method.
2. Background and Description of the Art
Flow cytometers are instruments which analyze biological cells and particles in a thin stream of fluid intersected by an illumination source, usually a laser beam, with the resulting forward and right angle scattered and fluorescent light analyzed with photomultiplier tubes (PMTs). Fluorescent channels are usually indicated by the designations F11, F12, F13, etc., depending on the number of channels in the instrument Each fluorescent channel is set with barrier filters to detect a selected specific dye while filtering out all others. The channel in which a specific dye is predominantly detectable may be referred to as its primary fluorescent channel while other fluorescent channels may be designated as secondary channels.
In order to obtain accurate and reproducible results, flow cytometers must be aligned and calibrated. When operation with more than one fluorescent dye, the instrument also requires compensation for the fluorescence PMTs. Alignment, compensation, and calibration ensure that the instrument will operate at its maximum efficiency, as well as achieving reproducibility such that data taken over time or with various instruments will be comparable.
Alignment is the process of adjusting and focusing the various optical and electrical components such that scatter and fluorescence signals are tuned to their highest intensity and tightest distribution, i.e., lowest coefficient of variation (CV) of the distribution. The components of the flow cytometer to be aligned include the laser, lenses, mirrors, barrier filters, and PMTs FIG. 1 shows what the dot plot of the forward and right angle scatter channels typically looks like when the instrument is not aligned The corresponding dot plot of FIG. 2 indicates proper alignment of those channels
Compensation is the process of electronically removing residual signals from fluorescent dyes in secondary fluorescence channels due to spectral overlaps not removed by the barrier filters for the respective channels When compensation circuits are turned off, the dot plot for the fluorescent channel F11 versus the fluorescent channel F12 appears as in FIG. 3, with fluorescent microbeads (2) and (3) overlapping the boundaries designated by blank microbeads (1). The dot plot of FIG. 4 shows that the compensation circuits are adjusted properly so that the intensity of any signal in the secondary fluorescent channels is equivalent to the blank samples. In other words, the dot group must be aligned with a blank or unlabeled sample in the secondary fluorescence channel. If this is not accomplished, the samples labeled with a single fluorescent dye will be counted in both fluorescent channels when it should only be counted in its respective primary fluorescent channel. In addition, if the compensation is set too high, as illustrated in FIG. 5, then data from the sample may be lost.
Multi-fluorescence analysis, i.e., analysis using two or more fluorescent dyes simultaneously, can be performed on a flow cytometer. However, to perform accurate analysis, it is necessary to adjust the electrical compensation circuits in the flow cytometer such that any fluorescence emission which overlaps into other fluorescence channels may be subtracted from such other channels As an example, using fluorescein and phycoerythrin as simultaneously employed fluorescent dyes, the green fluorescence channel for fluorescein may have a band pass emission filter of 520.+-.10 nanometers (nm) and the red fluorescence channel for phycoerythrin may have a band pass emission filter of 580.+-.10 nm. The emission spectra of fluorescein is such that part of its fluorescence will be seen in the phycoerythrin fluorescence channel of 580.+-.10 nm, and to a lesser degree, part of the emission of phycoerythrin will appear in the fluorescein fluorescence channel of 520.+-.10 nm.
A particular problem associated with numerous samples measured by flow cytometry relates to naturally occurring fluorescence, i.e., autofluorescence, of the sample. For example, a wide variety of biological cells contain naturally occurring fluorescent compounds such as riboflavin. Such autofluorescence introduces an additional complexity to the flow cytometer compensation process, and tends to promote mis-compensation (over- and/or under-compensation) in the respective fluorescence channels of the flow cytometer.
Calibration of a flow cytometer with proper standards ensures that the results from samples will be comparable over time and between different instruments. For the calibration of the intensity of fluorescence signals to be independent of the specific instrument and instrument settings, the excitation and emission spectra of the calibration standards and of the samples being measured must be equivalent and the measurements on each must be made under the same instrument settings. In addition, as described in U.S. Pat. Nos. 4,714,682; 4,767,206; and 4,774,189, and copending U.S. application Ser. No. 109,214, the disclosures of which hereby are incorporated by reference, when the calibration is made in terms of number of equivalent soluble fluorescent molecules, such correction factors as quenching and changes in extinction coefficient due to conjugation to other molecules, need not be taken into consideration.
Fluorescence calibration curves for flow cytometers may be constructed by plotting the mean or modal channels of the fluorescence intensity histograms of fluorescence microbead standards against the calibrated values of the number of equivalent soluble fluorescent dye molecules for the respective microbead standards, as shown in FIG. 6.
Small (0.1-2 microns) highly uniform microbeads are readily commercially available from a number of manufacturing companies, e.g., Seragen, Inc., Polysciences, Inc., and Interfacial Dynamics Corp. Production of large (2-50 microns) highly uniform microbeads are described in U.S. Pat. Nos. 4,247,434 and 4,336,173. The synthesis of fluorescent microbeads is taught in U.S. Pat. Nos. 4,157,323 and 4,179,685, but these microbeads are not intended to be used as uniform standards and their spectra are not designed to match those of labeled samples.
The aforementioned U.S. Pat. Nos. 4,714,682; 4,767,206; and 4,774,189, and copending application Ser. No. 109,214 relate to calibration of flow cytometers in terms of equivalent soluble fluorescent dye molecules with standards (calibration microbeads) that have matching fluorescent properties.
A kit of microbeads that match labeled cells is commercially available under the trademark CaliBrites from Becton Dickinson & Co. (Mountain View, California), which consists of three microbead populations: (i) an unlabeled population, (ii) a fluorescein-labeled population, and (iii) a phycoerythrin-labeled population. None of these microbeads are calibrated in any way, and they are intended only for alignment and compensation of a flow cytometer.
It is therefore an object of the present invention to provide a method for alignment, compensation, and/or calibration of a flow cytometer for analysis of selected samples which may comprise, in a specific aspect of the invention, naturally fluorescent samples), enabling the flow cytometer to operate at high efficiency with respect to fluorescence data generated thereby, and in a manner achieving reproducibility of data which is independent of the specific instrument and time-frame of the data measurement.
It is another object of the invention to provide a microbead standards kit for carrying out such alignment, compensation, and calibration method.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.